The importance of epigenetic transcriptional silencing of key tumor suppressor genes in many malignancies has been well established. The molecular mechanisms leading to this transcriptional silencing has recently begun to be understood over the last few years, leading to the concept that DNA methylation interacts in a dynamic way with nuclear histone modifications to either repress or enhance transcription. Pre-clinical work in chronic lymphocytic leukemia (CLL) by our chromatin remodeling team actively involved in this proposal has demonstrated these mechanisms of gene silencing is clinically relevant. Specifically, we have demonstrated marked variation in the amount of aberrant methylation in CLL patient samples ranging from 1% to 6% increase in specific gene methylation as compared to normal B cells. In addition, we have demonstrated that selected genes such as Dermis expressed-1 (DERMO-1), TWIST, and the metabotrobic glutamate receptor 7 (GRM7) are differentially methylated in primary tumor cells derived from patients with CLL. In addition, DERMO-1 and TWIST are associated with specific VH gene subtypes and prior treatment status. Furthermore, treatment of CLL cells with the hypomethylating agent decitabine promotes trapping of DNA methyltransferase 1 (DNMT1) on DNA and depletion of free DNMT1 protein with subsequent gene re-expression and caspase dependent apoptosis in CLL cells in vitro. Based upon this and our previous work with histone deacetylase inhibitors including valproic acid, we seek to test the overall hypothesis that application of epigenetic therapy targeting chromatin in CLL will relieve aberrant transcriptional repression of tumor suppressor genes, restore normal patterns of cell proliferation, differentiation and apoptosis and ultimately result in clinical benefit to patients with CLL. The specific aims of this proposal are: 1) to perform a minimal effective pharmacologic dose (MEPD) finding study of decitabine and then decitabine combined with valproic acid in fludarabine-refractory CLL patients, and 2) to perform the MEPD-directed studies concurrent with detailed pharmacologic and pharmacodynamic studies as part of the aim 1 clinical trial. This study will provide sufficient preliminary data to later pursue, as part of a separate application, a randomized phase II study to determine the clinical and gene re-expression efficacy of these two therapeutic approaches in fludarabine-refractory CLL. In addition, completion of this proposal will afford knowledge of the kinetics of DNMT1 inhibition in CLL and associated gene re-expression to allow pursuit of alternative combinations of epigenetically targeted therapies in the future.